Abstract
The young leaves of virescent mutants are yellowish and gradually turn green as the plants reach maturity. Understanding the genetic basis of virescent mutants can aid research of the regulatory mechanisms underlying chloroplast development and chlorophyll biosynthesis, as well as contribute to the application of virescent traits in crop breeding. In this study, fine mapping was employed, and a recessive gene (v 1) from a virescent mutant of Upland cotton was narrowed to an 84.1-Kb region containing ten candidate genes. The GhChlI gene encodes the cotton Mg-chelatase I subunit (CHLI) and was identified as the candidate gene for the virescent mutation using gene annotation. BLAST analysis showed that the GhChlI gene has two copies, Gh_A10G0282 and Gh_D10G0283. Sequence analysis indicated that the coding region (CDS) of GhChlI is 1269 bp in length, with three predicted exons and one non-synonymous nucleotide mutation (G1082A) in the third exon of Gh_D10G0283, with an amino acid (AA) substitution of arginine (R) to lysine (K). GhChlI-silenced TM-1 plants exhibited a lower GhChlI expression level, a lower chlorophyll content, and the virescent phenotype. Analysis of upstream regulatory elements and expression levels of GhChlI showed that the expression quantity of GhChlI may be normal, and with the development of the true leaf, the increase in the Gh_A10G0282 dosage may partially make up for the deficiency of Gh_D10G0283 in the v 1 mutant. Phylogenetic analysis and sequence alignment revealed that the protein sequence encoded by the third exon of GhChlI is highly conserved across diverse plant species, in which AA substitutions among the completely conserved residues frequently result in changes in leaf color in various species. These results suggest that the mutation (G1082A) within the GhChlI gene may cause a functional defect of the GhCHLI subunit and thus the virescent phenotype in the v1 mutant. The GhChlI mutation not only provides a tool for understanding the associations of CHLI protein function and the chlorophyll biosynthesis pathway but also has implications for cotton breeding.
Similar content being viewed by others
References
Archer EK, Ting BL (1996) A virescent plastid mutation in tobacco decreases peroxisome enzyme activities in seedlings. J Plant Physiol 149:520–526
Beale SI (1999) Enzymes of chlorophyll biosynthesis. Photosynth Res 60:43–73
Beale SI (2005) Green genes gleaned. Trends Plant Sci 10:309–312
Campbell BW et al (2015) Identical substitutions in magnesium chelatase paralogs result in chlorophyll-deficient soybean mutants. G3 Genes Genom Genet 5:123–131
Czarnecki O, Grimm B (2012) Post-translational control of tetrapyrrole biosynthesis in plants, algae, and cyanobacteria. J Exp Bot 63:1675–1687
Davey JW, Hohenlohe PA, Etter PD, Boone JQ, Catchen JM, Blaxter ML (2011) Genome-wide genetic marker discovery and genotyping using next-generation sequencing. Nat Rev Genet 12:499–510
Dong H et al (2013) A rice virescent-yellow leaf mutant reveals new insights into the role and assembly of plastid caseinolytic protease in higher plants. Plant Physiol 162:1867–1880
Duncan EN, Pate JB (1967) Inheritance and use of golden crown virescence in cotton: and its relationship to other virescent stocks. J Hered 58:237–239
Fodje MN, Hansson A, Hansson M, Olsen JG, Gough S, Willows RD, Al-Karadaghi S (2001) Interplay between an AAA module and an integrin I domain may regulate the function of magnesium chelatase. J Mol Biol 311:111–122
Gao M, Hu L, Li Y, Weng Y (2016) The chlorophyll-deficient golden leaf mutation in cucumber is due to a single nucleotide substitution in CsChlI for magnesium chelatase I subunit. Theor Appl Genet 129:1961–1973
Gibson LC, Willows RD, Kannangara CG, Von WD, Hunter CN (1995) Magnesium-protoporphyrin chelatase of Rhodobacter sphaeroides: reconstitution of activity by combining the products of the bchH, -I, and -D genes expressed in Escherichia coli. Proc Natl Acad Sci USA 92:1941–1944
Gibson LC, Jensen PE, Hunter CN (1999) Magnesium chelatase from Rhodobacter sphaeroides: initial characterization of the enzyme using purified subunits and evidence for a BchI-BchD complex. Biochem J 337:243–251
Gu Z, Huang C, Li F, Zhou X (2014) A versatile system for functional analysis of genes and microRNAs in cotton. Plant Biotechnol J 12:638–649
Hansson A, Kannangara CG, Von WD, Hansson M (1999) Molecular basis for semidominance of missense mutations in the XANTHA-H (42-kDa) subunit of magnesium chelatase. Proc Natl Acad Sci USA 96:1744–1749
He Z, Xie Z, Wang Y, Shen J, Li L (2013) Genetic analysis and breeding application of a novel rice mutant with virescent yellow leaves. Chin J Trop Crop 34:2145–2149
Hu F, Zhou Z (2006) Molecuar marker and genetic mapping of five mutant genes in upland cotton. Mol Plant Breed 4:680–684
Huang YS, Li HM (2009) Arabidopsis CHLI2 can substitute for CHLI1. Plant Physiol 150:636–645
Jensen PE, Gibson LC, Henningsen KW, Hunter CN (1996) Expression of the chlI, chlD, and chlH genes from the Cyanobacterium synechocystis PCC6803 in Escherichia coli and demonstration that the three cognate proteins are required for magnesium-protoporphyrin chelatase activity. J Biol Chem 271:16662–16667
Jensen PE, Gibson LC, Hunter CN (1998) Determinants of catalytic activity with the use of purified I, D and H subunits of the magnesium protoporphyrin IX chelatase from Synechocystis PCC6803. Biochem J 334:335–344
Jensen P, Gibson L, Hunter CN (1999) ATPase activity associated with the magnesium-protoporphyrin IX chelatase enzyme of Synechocystis PCC6803: evidence for ATP hydrolysis during Mg2+ insertion, and the MgATP-dependent interaction of the ChlI and ChlD subunits. Biochem J 339:127–134
Jones P et al (2014) InterProScan 5: genome-scale protein function classification. Bioinformatics 30:1236–1240
Karger GA, And JDR, Hunter CN (2001) Characterization of the binding of deuteroporphyrin IX to the magnesium chelatase H subunit and spectroscopic properties of the complex. Biochemistry 40:9291–9299
Killough DT, Horlacher WR (1933) The Inheritance of virescent yellow and red plant colors in cotton. Genetics 18:329–334
Kohel RJ (1973) Analysis of irradiation induced virescent mutants and the identification of a new virescent mutant (v5v5,v6v6) in Gossypium hirsutum L. Crop Sci 13:86–88
Kohel RJ (1974) Genetic analysis of a new virescent mutant in cotton. Crop Sci 14:525–527
Kohel RJ (1983) Genetic analysis of virescent mutants and the identification of virescents υ12, υ13, υ14, υ15 and υ16 υ17 in upland cotton. Crop Sci 23:289–291
Koncz C, Mayerhofer R, Konczkalman Z, Nawrath C, Reiss B, Redei GP, Schell J (1990) Isolation of a gene encoding a novel chloroplast protein by T-DNA tagging in Arabidopsis thaliana. Embo J 9:1337–1346
Lescot M et al (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 169:325–327
Li H et al (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25:2078–2079
Li F et al (2014) Genome sequence of the cultivated cotton Gossypium arboreum. Nat Genet 46:567–572
Li F et al (2015) Genome sequence of cultivated Upland cotton (Gossypium hirsutum TM-1) provides insights into genome evolution. Nat Biotechnol 33:524–530
Li Q et al (2016) Functional conservation and divergence of GmCHLI genes in polyploid soybean. Plant J 88:584–596
Liu X et al (2015) Gossypium barbadense genome sequence provides insight into the evolution of extra-long staple fiber and specialized metabolites. Sci Rep 5:14139
Lu C et al (2015) Development of chromosome-specific markers with high polymorphism for allotetraploid cotton based on genome-wide characterization of simple sequence repeats in diploid cottons (Gossypium arboreum L. and Gossypium raimondii Ulbrich). BMC Genom 16:1–12
Luo T et al (2013) Virus-induced gene silencing of pea CHLI and CHLD affects tetrapyrrole biosynthesis, chloroplast development and the primary metabolic network. Plant Physiol Bioch 65:17–26
Masuda T (2008) Recent overview of the Mg branch of the tetrapyrrole biosynthesis leading to chlorophylls. Photosynth Res 96:121–143
Meng LH, Wang RH, Zhu BZ, Zhu HL, Luo YB, Fu DQ (2016) Efficient virus-induced gene silencing in Solanum rostratum. PloS One 11:e0156228
Min L, He J, Xiao S, Zhang T, Pan J (1996) A Comprehensive report of studies on utilization of heterosis of virescent strains in upland cotton. Acta Gossypii Sin 8:113–119
Nagata N, Tanaka R, Satoh S, Tanaka A (2005) Identification of a vinyl reductase gene for chlorophyll synthesis in Arabidopsis thaliana and implications for the evolution of Prochlorococcus Species. Plant Cell 17:233–240
Percival AE, Kohel RJ (1974) Genetic analysis of virescent mutants in cotton. Crop Sci 14:439–440
Percival AE, Kohel RJ (1976) New virescent cotton mutant linked with the marker gene yellow petals. Crop Sci 16:503–505
Permingeat HR, Romagnoli MV, Sesma JI, Vallejos RH (1998) A simple method for isolating DNA of high yield and quality from cotton (shape Gossypium hirsutum L.) leaves. Plant Mol Biol Rep 16:89–89
Petersen BL, Moller MG, Jensen PE, Henningsen KW (1999) Identification of the Xan-g gene and expression of the Mg-chelatase encoding genes Xan-.f, -g and -h in mutant and wild type barley (Hordeum Vulgare L.). Hered 131:165–170
Qi Y et al (2015) A putative chloroplast thylakoid metalloprotease VIRESCENT3 regulates chloroplast development in Arabidopsis thaliana. J Biol Chem 291:3319–332
Quisenberry JE, Kohel RJ (1970) Genetics of the virescent-4 mutant in cotton. J Hered 5:212–214
Reid JD, Siebert CA, Bullough PA, Hunter CN (2003) The ATPase activity of the ChlI subunit of magnesium chelatase and formation of a heptameric AAA+ ring. Biochemistry 42:6912–6920
Rissler HM, Collakova E, DellaPenna D, Whelan J, Pogson BJ (2002) Chlorophyll biosynthesis. Expression of a second chl I gene of magnesium chelatase in Arabidopsis supports only limited chlorophyll synthesis. Plant Physiol 128:770–779
Rychlik W (2007) OLIGO 7 primer analysis software. Methods Mol Biol 402:35–60
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425
Sawers RJ et al (2006) The maize oil yellow1 (Oy1) gene encodes the I subunit of magnesium chelatase. Plant Mol Biol 60:95–106
Sheffield VC, Beck JS, Kwitek AE, Sandstrom DW, Stone EM (1993) The sensitivity of single-strand conformation polymorphism analysis for the detection of single base substitutions. Genomics 16:325–332
Soldatova O et al (2005) An Arabidopsis mutant that is resistant to the protoporphyrinogen oxidase inhibitor acifluorfen shows regulatory changes in tetrapyrrole biosynthesis. Mol Genet Genomics 273:311–318
Sonah H et al (2013) An improved genotyping by sequencing (GBS) approach offering increased versatility and efficiency of SNP discovery and genotyping. PloS One 8:e54603
Song M, Yang Z, Fan S, Zhu H, Pang C, Tian M, Yu S (2011) Physiological and biochemical analysis and identification of a short season cotton virescent mutant. Sci Agric Sin 44:3709–3720
Song M, Yang Z, Fan S, Zhu H, Pang C, Tian M, Yu S (2012) Cytological and genetic analysis of a virescent mutant in Upland cotton (Gossypium hirsutum L.). Euphytica 187:235–245
Sugimoto H, Kusumi K, Tozawa Y, Yazaki J, Kishimoto N, Kikuchi S, Iba K (2004) The virescent-2 mutation inhibits translation of plastid transcripts for the plastid genetic system at an early stage of chloroplast differentiation. Plant Cell Physiol 45:985–996
Tai PYP, Hammons RO, Matlock RS (1977) Genetic relationships among three chlorophyll-deficient mutants in peanut, Arachis hypogaea L. Theor Appl Genet 50:35–40
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729
Turcotte EL, Feaster CV (1973) The interaction of two genes for yellow foliage in cotton. J Hered 4:231–232
Turcotte EL, Percy RG (1988) Inhertance of a second virescent mutant in American PIMA cotton. Crop Sci 28:1018–1019
Vale RD (2000) AAA proteins. Lords of the ring. J Cell Biol 150:F13–F19
Walker CJ, Willows RD (1997) Mechanism and regulation of Mg-chelatase. Biochem J 327:321–333
Wang K et al (2012) The draft genome of a diploid cotton Gossypium raimondii. Nat Genet 44:1098–1103
Wang Q et al (2015a) Genome-wide mining, characterization, and development of microsatellite markers in Gossypium Sp. Sci Rep 5:10638
Wang S et al (2015b) Sequence-based ultra-dense genetic and physical maps reveal structural variations of allopolyploid cotton genomes. Genome Biol 16:108
Wendel JF, Cronn RC (2003) Polyploidy and the evolutionary history of cotton. Adv Agron 78:139–186
Xiao S, Huang J, Pan J, Zhang T (1996) Study on the utilization of heterosis between the virescent strain and the commercial cultivar in Upland cotton. Acta Gossypii Sin 8:71–76
Xing A et al (2014) A pair of homoeolog ClpP5 genes underlies a virescent yellow-like mutant and its modifier in maize. Plant J 79:192–205
You FM et al (2008) BatchPrimer3: a high throughput web application for PCR and sequencing primer design. BMC Bioinf 9:253
Yu S et al (2003) Genetics and breeding of cotton in China. Shangdong Science and Technology Press, Jinan
Zhang T, Pan J (1990) Allelic tests of virescent mutants and genetic identification of virescent V22 in Upland cotton. Jiangsu J Agric Sci 6:24–29
Zhang H et al (2006) Rice Chlorina-1 and Chlorina-9 encode ChlD and ChlI subunits of Mg-chelatase, a key enzyme for chlorophyll synthesis and chloroplast development. Plant Mol Biol 62:325–337
Zhang Q, Xue D, Li X, Long Y, Zeng X, Liu Y (2014) Characterization and molecular mapping of a new virescent mutant in rice. J Genet Genom 41:353–356
Zhang T et al (2015a) Sequencing of allotetraploid cotton (Gossypium hirsutum L. acc. TM-1) provides a resource for fiber improvement. Nat Biotechnol 33:531–537
Zhang H et al (2015b) A point mutation of magnesium chelatase OsCHLI gene dampens the interaction between CHLI and CHLD subunits in rice. Plant Mol Biol Rep 33:1975–1987
Acknowledgements
This research was funded by the National Natural Science Foundation of China (31621005).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Additional information
Communicated by S. Hohmann.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Mao, G., Ma, Q., Wei, H. et al. Fine mapping and candidate gene analysis of the virescent gene v 1 in Upland cotton (Gossypium hirsutum). Mol Genet Genomics 293, 249–264 (2018). https://doi.org/10.1007/s00438-017-1383-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00438-017-1383-4